The present invention relates to viral vectors comprising nucleic acid sequences coding for single chain interleukin-12 (single chain IL-12 or scIL-12) and a costimulator protein, and to the use of said vectors for gene therapy, especially for the treatment of tumors.
Tumors are still one of the most common causes of death of human beings living in industrialized countries. For example, the hepatocellular carcinoma (HCC) is a tumor with an average survival rate of 6 months after diagnosis of one or several larger tumors (Llovet J. M. et al., Hepatology, 1999, 29: 62-67). The currently used therapies, comprising radio frequency ablation, chemotherapy and percutaneous ethanol injection (PEI) result in some success, when treating smaller tumors, but they are insufficient to fight large tumors.
In the prior art, therefore it was suggested to treat HCC by means of gene therapy. Gene therapeutic treatments are based on the administration of a nucleic acid that is usually incorporated into the tumor cell, and has a sequence that destroys the tumor cell. To this end, a multitude of different strategies has been developed that will effect a destruction of tumor cells caused by the transferred nucleic acid sequences. An overview of such strategies for the treatment of HCC can be found in Ruiz et al., (Dig. Dis. 2001, 19:324-332). In this publication, the nucleic acids currently used in clinical trials for the treatment of HCC in humans, are categorized into one of the four following groups, according to the treatment strategy:
(1) Transfer of Tumor Suppressor Genes:
                This strategy is based on the fact that the nucleic acid used for gene therapy contains a gene, which encodes a gene product that inhibits growth of the tumor or that induces apoptosis in the tumor cells. Most clinical trials are based on the transfer of the p53 gene.(2) Therapy Using Immune Genes        This strategy is based on the fact that the nucleic acids used for gene therapy comprises sequences encoding gene products that stimulate the patient's immune system and induce an immune response directed against the tumor cells. The immune response itself finally results in the destruction of the tumor. Numerous cytokines, costimulator molecules and tumor-specific molecules have been suggested for the use in a therapy using immune genes.(3) Therapy Using Suicide Genes        In this procedure, the nucleic acid used for gene therapy encodes a gene product, for example an enzyme, which transforms a non-toxic compound into an agent that is cytotoxic for the tumor cell.(4) Transfer of Oncolytic Viruses        In this variation of gene therapy, nucleic acid vectors are used that are based on viral sequences. Vectors with oncolytic viral sequences have a tumor-specific promoter that regulates the replication of the virus, thus enabling selective viral growth in the tumor cells.        
During the therapy using immune genes (also called immunotherapy) that is relevant for the current application, nucleic acids are administered that comprise sequences, which activate the immune system and are directed against the tumor. As a general property, the immune system does not only recognize antigens but also tumor-specific structures on tumor cells. Therefore, the activation of the immune system can result in the destruction of the tumor caused by components of the immune system.
According to prior art, numerous molecules are known that stimulate the immune system or modulate an immune reaction, in particular the cytokines. It was recognized very early that cytokines also have anti-tumor activities. For example, it was reported that IL-12 is a stimulator of the cellular immunity and that it exhibits strong anti-tumor activity (Brunda et al., J. Exp. Med. 1993, 178: 1223-1230). However, the administration of the recombinant IL-12 protein itself as an anti-tumor-agent failed due to the toxic side-effects of the cytokine when used in therapeutically relevant doses (Lotze et al., Ann. N.Y. Acad. Sci., 1997, 795: 440-454; and Cohen J., Science, 1995, 270: 908).
Therefore, it was suggested to introduce a nucleic acid encoding a cytokine into the tumor, thus enabling a local activation of the immune system. Hock et al. (Proc. Natl. Acad. Sci. USA, 1993, 90: 2774-2778) for example, describe the transfer of the interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), TNF or IFN-γ gene into tumor cell lines and the use of these tumor cell lines for the induction of tumors in animals. All transgenic tumor cell lines generated a rejection reaction against the tumor cells. Dependent on the cytokine used, different cell types of the immune system of the laboratory animals were involved in this rejection reaction (CD4+, CD8+, CD3+).
Vectors coding for IL-12 were also tested for their suitability in immunotherapy. IL-12, also known as CMLF (cytotoxic lymphocyte maturation factor) or NKSF (natural killer cell stimulatory factor), is a heterodimeric cytokine, which is naturally produced by activated peripheral B-lymphocytes. The protein consists of two subunits with relative molecular weights of 40 and 35 kDa, respectively, that are covalently linked by disulfide bridges. The disulfide bridges are essential for the biological activity. As already indicated by the different names, the protein stimulates the proliferation of activated human lyphoblasts and activates natural killer cells.
Vectors coding for the different subunits of this protein were used for the treatment of tumors (Barajas et al., Hepatology, 2001, 33: 52-61; Mazzolini et al., Cancer Gene Therapy, 1999, 6: 514-522). Furthermore, these vectors were used in combination with other sequences for immunotherapy. In particular, they were used in combination with sequences coding for a costimulator protein, contained within the same or in a different vector, for the treatment of tumors (Gyorffy et al., J. Immunology, 2001, 166: 6212-6217; Martinet et al., Gene Therapy, 2002, 9: 786-792; Martinet et al., Journal of National Cancer Institute, 2000, 92: 931-936; Guinn et al., J. Immunology, 1999, 162: 5003-5010; and Emtage et al., J. Immunology, 1998, 160: 2531-2538).
Further, IL-12 has already been expressed as single chain IL-12, yielding good activity, such as a protein comprising the different subunits linked together in one fusion protein (Lieschke et al., Nature Biotechnology, 1997, 15: 35-40). In a different therapeutic procedure it was suggested to remove tumor cells from the patient and treat these cells in vitro with a plasmid coding for single chain IL-12 or IL-12 and a costimulator (US 2002/0018767). Following this in vitro treatment the tumor cells shall be re-implanted into the patient. This procedure, therefore, comprises several operations on the patient and a re-implantation of tumor cells into the patient, which is likely to prevent many patients from undergoing such a treatment.
None of the previously used nucleic acids could prevail in the treatment of mammals, preferably in the treatment of humans. Although, for example, the publication by Ruiz et al (loc. cit.) described treatment procedures involving a very high dosage of the vector used for therapy (3×109−2.5×1013 plaque forming units, PFU, per dose), it was observed that none or only negative results were obtained in the corresponding clinical trials. However, especially the dose of the nucleic acids is a critical factor in gene therapy, because negative side effects or the release of the vector from the tumor are expected when doses are used that are too high.